Ex vivo cultured human hepatocytes serve as highly useful models of human liver in studies of metabolism and toxicology. One of the first metabolic safety tests for a lead compound in the pharmaceutical industry is the examination of which liver enzymes are affected by its presence, an assay system that relies solely upon the availability of fresh primary (1o) hepatocytes. While these assays play a critical role in the drug development and approval process, there are two major obstacles: cell supply and maintenance of cell function ex vivo. This shortage has motivated the exploration of stem-cell derived hepatocytes generated from human embryonic stem (hES) cells or induced pluripotent stem (iPS) cells. In theory, these cell lines could provide a consistent and renewable source of hepatocytes. However, reproducible protocols to differentiate these cells into hepatocytes that acquire/ maintain mature hepatic functions are lacking. Although hES cells can be subjected to liver-specific differentiation regimens and acquire measurable expression of cytochrome P450 enzymes, levels of enzymatic activity are significantly lower than those in freshly isolated adult human hepatocytes. Thus, new strategies are needed that either unlock the replicative potential of 1o hepatocytes in vitro or render stem cell-derived hepatocytes functionally equivalent to 1o human hepatocytes. Recently, increased success has been achieved when culturing 1o cells in 3-dimensions (3D) as they typically retain more liver-associated functions. Organovo's 3D proprietary bioprinting platform is perfectly positioned to take full advantage of these findings b leveraging an automated precision instrument to build small-scale liver tissues layer-by-layer in vitro using micro-scale building blocks ('bio-ink') comprised of liver cells. Application of the technology to the in vitro engineering of cardiac tissue and blood vessels has shown that 3D bioprinted tissues generated by this platform mature in vitro to mimic the architecture of the native tissue in vivo, including the in situ deposition of native tissue-like matrix and the formaton of microvasculature. In the aims proposed herein, our goal is to extend the platform to generate in vitro human liver surrogates, first using 1o hepatocytes and then liver origin iPS-derived hepatocytes. Initially, we will develop the methods to generate and print liver bio-ink, to achieve the Milestone of bioprinting a 3D human liver tissue that remains viable and functional for at least 3-5 days in an in vitro culture system. Success in Aim 1 alone will lead to the development of a commercializable 1o hepatocyte-based tissue model, although multi-well product availability will be limited by hepatocyte supply. To address the shortage of 1o human hepatocytes, the second goal will be to substitute iPS-derived hepatocytes as a renewable cell source, leveraging the 3D conformation to foster more complete differentiation/maturation and achieve Milestone 2. The 3D human liver surrogates generated by this proposal will not only provide a valuable, commercializable tool for drug discovery and development, but can be extended to develop specific 3D human models of liver disease and implantable constructs for therapeutic augmentation of liver function in vivo. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop small-scale living 3-dimensional (3D) liver tissues, using a renewable source of functional liver cells, in a format compatible with current methods. Organovo's patent-pending bioprinting platform enables fabrication of 3D human tissues using cells as building blocks, without the use of synthetic or native tissue-derived biomaterials or scaffolding, yielding a tissue in vitro that recapitulates native tissue architecture and biology. The human liver tissues generated by this project have the potential to substantially improve the drug development process by providing a superior model of liver biology and liver toxicology. This effort could greatly reduce the cost of drug development by reducing the number of late stage drug failures thus the overall cost of drugs as well.